CN113684237A - 一种具有免疫调节活性的螺旋藻小分子肽的制备方法 - Google Patents
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Abstract
本发明公开了一种具有免疫调节活性的螺旋藻小分子肽的制备方法,涉及生物技术领域。本发明是采用钝顶螺旋藻干粉为原料,依次经过分步酶解、过滤脱渣、脱色脱腥、超滤、纳滤膜脱盐浓缩、离心分离、灭菌、离心喷雾干燥后获得成品。本发明所制备的螺旋藻小分子肽能够促进器官的发育、改善机体免疫细胞状态、调节体内免疫活性物质的分泌表达,并提高机体的免疫能力。
Description
技术领域
本发明属于生物技术领域,具体涉及一种具有免疫调节活性的螺旋藻小分子肽的制备方法。
背景技术
螺旋藻素有“微型绿色功能性营养宝库”的美誉,其富含大量的优质可溶性蛋白。随着人类基因组序列分析和功能蛋白组学工作的进一步展开,越来越多的科学研究表明,在生命活动中(如分子识别、信号转导、细胞分化及个体发育等)起决定作用的并非是那些分子量大、结构复杂的蛋白质,而是那些结构相对简单的中、小分子物质—生物活性多肽,这些小分子生物活性多肽极易被人体快速和多途径地吸收,营养并无损失,而且产生特定的生理功效。
钝顶螺旋藻是螺旋藻中的优良品种,其蛋白质含量最高,藻蓝蛋白含量最多,SOD活性高,可溶性蛋白量高。钝顶螺旋藻能够提高机体免疫力、促进肠胃蠕动。因此利用钝顶螺旋藻经过蛋白酶的水解得到螺旋藻小分子肽,可以使其营养成分更好被人体吸收利用,可应用于食品、保健品及医药产品领域。
发明内容
基于此,本发明提供了一种具有免疫调节活性的螺旋藻小分子肽的制备方法,其是以钝顶螺旋藻干粉为原料,经过分步酶解、过滤等处理后获得螺旋藻小分子肽,并经小鼠功效实验验证具有免疫调节作用。
本发明具有免疫调节活性的螺旋藻小分子肽的制备方法包括以下步骤:
(1)原料选择:选择钝顶螺旋藻干粉为原料;
(2)配制螺旋藻溶液:钝顶螺旋藻干粉中加入水,按料液比为1:10进行混合,并升温至52-55℃,搅拌1h,得到螺旋藻溶液;
(3)分步酶解:在所述螺旋藻溶液中加入占螺旋藻干粉重量0.1%-0.15%的纤维素酶和占螺旋藻干粉重量0.1%-0.15%的果胶酶,充分酶解2h,再加入占螺旋藻干粉重量0.1%-0.2%的复合蛋白酶和占螺旋藻干粉重量0.05%-0.1%的木聚糖酶进行酶解1-2h,酶解结束后灭酶;
(4)过滤脱渣:经灭酶处理后的酶解液进行过滤,获得清液;
(5)脱色脱腥:将步骤(4)得到的清液经过活性炭纤维膜设备进行脱色脱腥处理,过滤后获得脱色脱腥液;
(6)超滤:将脱色脱腥液采用超滤膜进行超滤以去除杂质,获得超滤液;
(7)纳滤膜脱盐浓缩:将超滤液经纳滤膜进行浓缩、脱盐处理,获得浓缩液,所述浓缩液的浓度达到12%-15%,灰分≤1%,脱盐率达到95%以上;
(8)离心分离:将浓缩液进行离心分离处理,以达到料液的高度纯化;
(9)灭菌:将离心分离得到的清液进行灭菌处理;
(10)离心喷雾干燥,将灭菌后的清液采用高速离心喷雾干燥粒机进行喷粉干燥;
(11)成品包装。
优选的,步骤(3)所述的复合蛋白酶是由南宁东恒华道生物科技有限责任公司生产,其是由内切蛋白酶、外切蛋白酶和风味蛋白酶组成。
优选的,步骤(4)采用单层正压过滤器对酶解液进行过滤。
优选的,步骤(8)采用高速管式分离机对浓缩液进行离心分离。
更优选的,步骤(8)离心处理的转速为16000r/min,分离因数为:15050R.C.F
优选的,步骤(9)采用列管式高温瞬时灭菌设备进行灭菌处理,所述灭菌设备设置有预热装置、灭菌装置、热回收装置和冷却装置。
与现有技术相比,本发明具有以下有益效果:
本发明所制备的螺旋藻小分子肽粉呈黄绿色,溶解速度快,溶解后溶液澄清透明,无任何杂质且分子量相对集中且<1000Da,蛋白含量(以干基计)>70%,低聚肽含量(以干基计)>60%,可应用于食品、保健品及医药产品中。
通过对免疫低下小鼠体重增长率、免疫器官指数、血清白介素水平及免疫球蛋白的影响,研究螺旋藻小分子肽的免疫调节作用。实验结果表明:螺旋藻小分子肽能提高环磷酰胺诱导免疫能力低下小鼠的体重生长率、免疫器官指数和改善脾脏病理学变化;能提高免疫能力低下小鼠的细胞因子(IL-1α、IL-6、IFN-γ)和免疫球蛋白(IgG、IgM)的水平。本发明螺旋藻小分子肽可以通过促进免疫能力低下小鼠的中枢和外周免疫器官的发育、改善机体免疫细胞状态、调节体内免疫活性物质的分泌表达,提高机体免疫能力,发挥其免疫活性。
附图说明
图1为本发明制备螺旋藻小分子肽的工艺流程图。
具体实施方式
下面结合具体实施例对本发明作进一步说明。
实施例1
具有免疫调节活性的螺旋藻小分子肽的制备方法包括以下步骤:
(1)原料选择:选择钝顶螺旋藻干粉为原料;
(2)配制螺旋藻溶液:钝顶螺旋藻干粉中加入水,按料液比为1:10进行混合,并升温至54℃,搅拌1h,得到螺旋藻溶液;
(3)分步酶解:在所述螺旋藻溶液中加入占螺旋藻干粉重量0.1%-0.15%的纤维素酶和占螺旋藻干粉重量0.1%-0.15%的果胶酶,充分酶解2h,再加入占螺旋藻干粉重量0.1%-0.2%的复合蛋白酶和占螺旋藻干粉重量0.05%-0.1%的木聚糖酶进行酶解1-2h,酶解结束后85℃灭酶20min;
(4)过滤脱渣:经灭酶处理后的酶解液通过单层正压过滤器进行脱渣,把酶解液中的螺旋藻残渣和液体进行完全分离,过滤温度55℃,工作压力0.3MPa,获得清液;
(5)脱色脱腥:将步骤(4)得到的清液降温至50℃后,经过活性炭纤维膜设备进行脱色脱腥处理,过滤后获得脱色脱腥液;
(6)超滤:将脱色脱腥液采用超滤膜进行超滤以去除杂质,过滤温度60℃,过滤压力0.2MPa,获得超滤液;
(7)纳滤膜脱盐浓缩:将超滤液经纳滤膜进行浓缩、脱盐处理,获得浓缩液,所述浓缩液的浓度达到12%,灰分≤1%,脱盐率达到95%以上,所述浓缩、脱盐运行压力1.5MPa,运行温度35℃;
(8)离心分离:将浓缩液采用高速管式分离机进行离心分离处理,以达到料液的高度纯化,其中分离筒转速为16000r/min,分离因数为:15050R.C.F;
(9)灭菌:将离心分离得到的清液采用列管式高温瞬时灭菌设备灭菌处理,杀菌温度:120℃,出料温度50℃;
(10)离心喷雾干燥,将灭菌后的清液采用高速离心喷雾干燥粒机进行喷粉干燥,进风口温度190℃,出风口温度97℃;
(11)成品包装。
实验部分
对本发明得到的螺旋藻小分子肽的性能进行检测,具体方法如下:
选用SPF级ICR健康雌性小鼠60只,初始体重18~22g,饲养于屏障环境,温度范围为(22±2)℃,相对湿度为50%~60%,明暗交替时间为12h∶12h,自由进食、饮水。实验动物适应性喂养1d,正式实验第1天称量小鼠初始体重,并按体重随机分为5组,每组12只,其中一组为正常对照组,另外四组小鼠均采用隔天皮下注射环磷酰胺制造免疫功能低下小鼠模型,即为环磷酰胺模型组,对三组环磷酰胺模型组分别注射不同剂量的螺旋藻小分子肽,获得螺旋藻小分子肽低剂量组、螺旋藻小分子肽中剂量组、螺旋藻小分子肽高剂量组。其中螺旋藻小分子肽低剂量组中注射螺旋藻小分子肽量为0.125mg/g;螺旋藻小分子肽中剂量组中注射螺旋藻小分子肽量为0.500mg/g;螺旋藻小分子肽高剂量组中注射螺旋藻小分子肽量为2.000mg/g。
结果与讨论
1、通过免疫器官、免疫活性物质两方面探讨螺旋藻小分子肽对免疫能力低下小鼠的免疫调节作用。螺旋藻小分子肽对免疫能力低下小鼠体重增长率的影响结果见表1。
表1
组别 | 只数 | 初体重/g | 终体重/g | 体重增长率/% |
正常对照组 | 12 | 19.62±0.76 | 23.52±0.47 | 19.80% |
环磷酰胺模型组 | 12 | 19.85±0.28 | 21.17±0.32 | 6.74% |
低剂量组 | 12 | 20.15±0.72 | 22.79±0.67 | 13.10% |
中剂量组 | 12 | 19.81±0.68 | 23.10±0.40 | 16.67% |
高剂量组 | 12 | 20.01±0.29 | 23.53±0.61 | 17.59% |
体重增长率能直接或间接反映出机体健康状况。小鼠体重增长率的下降也是免疫能力低下模型造模成功的标志。由表1可知,与正常组相比,其他组别的体重增长率均有不同程度的下降,这表明免疫能力低下小鼠模型构建成功;螺旋藻小分子肽低、中、高剂量组较模型组的体重增长率均有所上升,且呈现量效关系。这说明螺旋藻小分子肽对环磷酰胺所导致的体重下降具有一定的恢复作用,可以改善免疫能力低下引起的体重下降。
2、螺旋藻小分子肽对小鼠免疫器官指数的影响结果见表2。
表2
组别 | 胸腺指数/(mg/g) | 脾脏指数/(mg/g) |
正常对照组 | 4.01±0.12# | 5.71±0.34# |
环磷酰胺模型组 | 2.68±0.45* | 4.08±0.37* |
低剂量组 | 3.82±0.22* | 4.89±0.19# |
中剂量组 | 3.17±0.22*# | 4.68±0.47# |
高剂量组 | 3.02±0.13# | 4.47±0.48# |
注:*为与空白对照组差异显著,且P<0.05;#为与模型组差异显著,且P<0.05(下同)
机体免疫器官的发育和状态直接决定着免疫***的应答能力。目前,免疫器官指数是反映和评价机体免疫***和免疫状态的重要指标之一。由表2可知,与正常组相比,模型组的胸腺指数和脾脏指数显著下降,说明环磷酰胺能抑制小鼠免疫器官的发育并诱发免疫器官发生萎缩;与模型对照组相比,螺旋藻小分子肽低、中、高剂量组的小鼠胸腺指数和脾脏指数均有不同程度的改善,且呈现量效关系,螺旋藻小分子肽高剂量组和正常组的胸腺指数并无显著性差异(P>0.05)。这表明螺旋藻小分子肽能明显缓解环磷酰胺构建的免疫能力低下小鼠免疫器官的萎缩,并促进其发育,增强免疫能力。
3、螺旋藻小分子肽对免疫能力低下小鼠血清白介素水平的影响结果见表3。
表3
组别 | 只数 | IL-1α/(pg/mL) | IL-6/(pg/mL) | IFN-γ/(pg/mL) |
正常对照组 | 12 | 30.11±0.54# | 36.08±0.56# | 23.58±0.57# |
环磷酰胺模型组 | 12 | 23.58±2.75* | 28.81±1.85* | 15.56±1.15* |
低剂量组 | 12 | 26.74±0.84*# | 30.70±1.04* | 16.99±1.50* |
中剂量组 | 12 | 28.86±0.96# | 33.01±1.12*# | 18.47±0.91*# |
高剂量组 | 12 | 29.78±0.21# | 32.86±1.88*# | 21.03±1.26*# |
IL-1α是IL-1家族中的典型代表之一,又称淋巴细胞活化因子,在免疫和炎症反应及机体生长代谢方面有着不可或缺的功能。IL-6是由机体多种免疫细胞产生的一种糖蛋白,主要作用的靶细胞是B淋巴细胞,又称为B细胞刺激因子。IFN-γ是特异性免疫的调节剂,能诱导Th0型细胞分化成Th1型细胞,并且能激活巨噬细胞、细胞毒性T细胞和清除胞内病原体。由表3可知,与正常组相比,模型对照组的血清IL-1α、IL-6、IFN-γ均显著下降(P<0.05),这表明环磷酰胺可引起机体白介素水平的显著下降,降低机体免疫防御能力;与模型对照组相比,螺旋藻小分子肽中、高剂量组可以显著提升IL-1α、IL-6、IFN-γ水平(P<0.05),且中、高剂量组IL-1α和正常组无显著性差异(P>0.05)。结果表明螺旋藻小分子肽可以显著缓解环磷酰胺导致的免疫能力低下小鼠体内白介素水平的降低,且呈现量效关系。
4、螺旋藻小分子肽对免疫能力低下小鼠免疫球蛋白的影响结果见表4
表4
组别 | IgG/(pg/mL) | IgM/(pg/mL) |
正常对照组 | 11.80±0.39# | 1.86±0.10# |
环磷酰胺模型组 | 7.67±0.61* | 1.16±0.05* |
低剂量组 | 8.68±1.16* | 1.37±0.18* |
中剂量组 | 10.45±1.09# | 1.66±0.10# |
高剂量组 | 11.54±0.71# | 1.74±0.12# |
IgG是体液免疫应答所产生的主要抗体,约占血清中抗体总量的80%,具有抗菌、中和病毒及免疫调节等生物活性。IgM是机体初次免疫应答中分泌最早的抗体,在抵御原发性感染的过程中发挥着重要作用。由表4可知,与正常组相比,模型对照组的IgG水平显著下降(P<0.05),这说明环磷酰胺可以抑制机体IgG、IgM的分泌和表达;与模型对照组相比,螺旋藻小分子肽中、高剂量组可以显著提升IgG、IgM水平(P<0.05),且螺旋藻小分子肽中、高剂量组IgM水平和正常组无显著性差异(P>0.05)。结果表明螺旋藻小分子肽可以显著缓解环磷酰胺导致的免疫能力低下小鼠体内免疫球蛋白水平的降低,且呈现量效关系。
需要说明的是,以上列举的仅是本发明的若干个具体实施例,显然本发明不仅仅限于以上实施例,还可以有其他变形。本领域的技术人员从本发明公开内容直接导出或间接引申的所有变形,均应认为是本发明的保护范围。
Claims (6)
1.一种具有免疫调节活性的螺旋藻小分子肽的制备方法,其特征在于,包括以下步骤:
(1)原料选择:选择钝顶螺旋藻干粉为原料;
(2)配制螺旋藻溶液:钝顶螺旋藻干粉中加入水,按料液比为1:10进行混合,并升温至52-55℃,搅拌1h,得到螺旋藻溶液;
(3)分步酶解:在所述螺旋藻溶液中加入占螺旋藻干粉重量0.1%-0.15%的纤维素酶和占螺旋藻干粉重量0.1%-0.15%的果胶酶,充分酶解2h,再加入占螺旋藻干粉重量0.1%-0.2%的复合蛋白酶和占螺旋藻干粉重量0.05%-0.1%的木聚糖酶进行酶解1-2h,酶解结束后灭酶;
(4)过滤脱渣:经灭酶处理后的酶解液进行过滤,获得清液;
(5)脱色脱腥:将步骤(4)得到的清液经过活性炭纤维膜设备进行脱色脱腥处理,过滤后获得脱色脱腥液;
(6)超滤:将脱色脱腥液采用超滤膜进行超滤以去除杂质,获得超滤液;
(7)纳滤膜脱盐浓缩:将超滤液经纳滤膜进行浓缩、脱盐处理,获得浓缩液,所述浓缩液的浓度达到12%-15%,灰分≤1%,脱盐率达到95%以上;
(8)离心分离:将浓缩液进行离心分离处理,以达到料液的高度纯化;
(9)灭菌:将离心分离得到的清液进行灭菌处理;
(10)离心喷雾干燥,将灭菌后的清液采用高速离心喷雾干燥粒机进行喷粉干燥;
(11)成品包装。
2.根据权利要求1所述具有免疫调节活性的螺旋藻小分子肽的制备方法,其特征在于,步骤(3)所述的复合蛋白酶是由内切蛋白酶、外切蛋白酶和风味蛋白酶组成。
3.根据权利要求1所述具有免疫调节活性的螺旋藻小分子肽的制备方法,其特征在于,步骤(4)采用单层正压过滤器对酶解液进行过滤。
4.根据权利要求1所述具有免疫调节活性的螺旋藻小分子肽的制备方法,其特征在于,步骤(8)采用高速管式分离机对浓缩液进行离心分离。
5.根据权利要求4所述具有免疫调节活性的螺旋藻小分子肽的制备方法,其特征在于,步骤(8)离心处理的转速为16000r/min,分离因数为:15050R.C.F 。
6.根据权利要求1所述具有免疫调节活性的螺旋藻小分子肽的制备方法,其特征在于,步骤(9)采用列管式高温瞬时灭菌设备进行灭菌处理,所述灭菌设备设置有预热装置、灭菌装置、热回收装置和冷却装置。
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CN113355383A (zh) * | 2021-06-04 | 2021-09-07 | 中国水产科学研究院黄海水产研究所 | 一种螺旋藻综合利用加工方法 |
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CN108753874A (zh) * | 2018-06-21 | 2018-11-06 | 哈尔滨华藻生物科技开发有限公司 | 一种小分子活性肽新型螺旋藻粉的制备方法 |
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